There is a fundamental gap in understanding how several components of engineered gene-editing nucleases achieve gene modification in human cells. Continued existence of this gap represents an important problem because, until it is filled, use of genome surgery tools will be limited, as it is not clear why various nucleases fail and why some succeed in producing desired gene edits. The long-term goal is to watch genome surgery in action to understand the bottlenecks in performing genome surgery on human cells in vitro with precisely controlled gene-editing particles, comprised of CRISPR-Cas9 components. Particles will be systematically assembled with various components and delivered in a controlled fashion to patient-derived cells and tissues. Live, in situ high content imaging and analysis within customized cell substrates will monitor genome surgery. These capabilities will explore large sequence variation of CRISPR-Cas9 components along with new assemblies of CRISPR-Cas9 components. The central hypothesis is that new assemblies of CRISPR-Cas9 particles can probe different biological processes of trafficking, DNA-double strand break formation and DNA repair involved in genome surgery. This hypothesis will be tested with respect to generating two types of gene edits involving non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways at several genomic loci within patient-derived stem cells and tissues. An overarching rationale for the proposed research programs is that robust gene editing techniques could enable the production of personalized drugs, cell therapies and gene therapies for future genomic and precision medicine. Guided by strong preliminary data, this hypothesis will be tested by pursuing three research programs: 1) Assemble Cas9 particles to identify biological processes that promote reporter-less transcript tagging of stem cell fate in culture; 2) Assemble Cas9 particles to identify biological processes that promote gene correction of diseased mutations in stem cells; and, 3) Assemble Cas9 particles to identify biological processes that promote gene correction of diseased mutations in microtissues. Under the first research program, an already proven platform, to assemble hundreds of unique Cas9 particles and edit patient-derived cells in a multiplexed manner, will be used to monitor the production of small gene edits by NHEJ within stem cell marker genes. Under the second and third research programs, this platform will be applied to gene-correct diseased mutations via HDR in induced pluripotent stem cells and microtissues matured from them. The approach is innovative, in the applicant's opinion, because it departs from the status quo by systematically changing multiple components at a time using novel methods in patient-derived cells. The proposed research is significant, because it is expected to advance and expand understanding of how genome surgery tools can be applied for the generation of advanced therapeutics, ranging from targeted small molecules to autologous cell therapies. Ultimately, such knowledge has the potential to set the foundation for new preclinical platforms in Precision Medicine.